Biological products

ABSTRACT

There is disclosed antibody molecules containing at least one CDR derived from a mouse monoclonal antibody having specificity for human CD22. There is also disclosed a CDR grafted antibody wherein at least one of the CDRs is a modified CDR. Further disclosed are DNA sequences encoding the chains of the antibody molecules, vectors, transformed host cells and uses of the antibody molecules in the treatment of diseases mediated by cells expressing CD22.

This application is a Continuation of application Ser. No. 15/417,973,filed on Jan. 27, 2017, which is a Continuation of application Ser. No.15/069,078, filed on Mar. 14, 2016, which is a Continuation ofapplication Ser. No. 11/519,585, filed on Sep. 11, 2006, which is aContinuation of application Ser. No. 10/428,408, filed May 2, 2003, nowU.S. Pat. No. 7,355,011, which claims priority under 35 U.S.C. §119(a)-(d) to United Kingdom Application No. GB 0210121.0, filed May 2,2002, all applications being incorporated by reference herein in theirentireties.

The present invention relates to an antibody molecule having specificityfor antigenic determinants of the B lymphocyte antigen, CD22. Thepresent invention also relates to the therapeutic uses of the antibodymolecule and methods for producing the antibody molecule.

In a natural antibody molecule, there are two heavy chains and two lightchains. Each heavy chain and each light chain has at its N-terminal enda variable domain. Each variable domain is composed of four frameworkregions (FRs) alternating with three complementarity determining regions(CDRs). The residues in the variable domains are conventionally numberedaccording to a system devised by Kabat et al. This system is set forthin Kabat et al., 1987, in Sequences of Proteins of ImmunologicalInterest, US Department of Health and Human Services, NIH, USA(hereafter “Kabat et al. (supra)”). This numbering system is used in thepresent specification except where otherwise indicated.

The Kabat residue designations do not always correspond directly withthe linear numbering of the amino acid residues. The actual linear aminoacid sequence may contain fewer or additional amino acids than in thestrict Kabat numbering corresponding to a shortening of, or insertioninto, a structural component, whether framework or CDR, of the basicvariable domain structure. The correct Kabat numbering of residues maybe determined for a given antibody by alignment of residues of homologyin the sequence of the antibody with a “standard” Kabat numberedsequence.

The CDRs of the heavy chain variable domain are located at residues31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3)according to the Kabat numbering.

The CDRs of the light chain variable domain are located at residues24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3)according to the Kabat numbering.

Construction of CDR-grafted antibodies is described in European PatentApplication EP-A-0239400, which discloses a process in which the CDRs ofa mouse monoclonal antibody are grafted onto the framework regions ofthe variable domains of a human immunoglobulin by site directedmutagenesis using long oligonucleotides. The CDRs determine the antigenbinding specificity of antibodies and are relatively short peptidesequences carried on the framework regions of the variable domains.

The earliest work on humanising monoclonal antibodies by CDR-graftingwas carried out on monoclonal antibodies recognising synthetic antigens,such as NP. However, examples in which a mouse monoclonal antibodyrecognising lysozyme and a rat monoclonal antibody recognising anantigen on human T-cells were humanised by CDR-grafting have beendescribed by Verhoeyen et al. (Science, 239, 1534-1536, 1988) andRiechmann et al. (Nature, 332, 323-324, 1988), respectively.

Riechmann et al., found that the transfer of the CDRs alone (as definedby Kabat (Kabat et al. (supra) and Wu et al., J. Exp. Med., 132,211-250, 1970)) was not sufficient to provide satisfactory antigenbinding activity in the CDR-grafted product. It was found that a numberof framework residues have to be altered so that they correspond tothose of the donor framework region. Proposed criteria for selectingwhich framework residues need to be altered are described inInternational Patent Application No. WO 90/07861.

A number of reviews discussing CDR-grafted antibodies have beenpublished, including Vaughan et al. (Nature Biotechnology, 16, 535-539,1998).

Malignant lymphomas are a diverse group of neoplasms. The majority ofcases occur in older people. Non-Hodgkins Lymphoma (NHL) is a diseasethat currently affects 200,000 to 250,000 patients in the U.S. It is thesecond fastest rising cancer in the U.S., rising at a rate of about55,000 new cases per year. The incidence is rising at a rate that isgreater than can be accounted for simply by the increasing age of thepopulation and exposure to known risk factors.

The classification of lymphoma is complex, and has evolved in recentdecades. In 1994 the Revised European-American Lymphoma (REAL)classification was introduced. This classification organises lymphomasof B cell (the most frequently identified), T cell and unclassifiableorigin into agreed subtypes. In everyday practice, the grouping of NHLsinto low, intermediate and high-grade categories on the basis of theirgeneral histological appearance, broadly reflects their clinicalbehaviour.

NHL predominantly affects the lymph nodes but, in individual patients,the tumour may involve other anatomical sites such as the liver, spleen,bone marrow, lung, gut and skin. The disease commonly presents as apainless enlargement of lymph nodes. Extranodal lymphoma most frequentlyaffects the gut, although primary lymphoma of virtually every organ hasbeen documented. Systemic symptoms include fever, sweats, tiredness andweight loss.

Until recently, the Ann Arbor staging system, based entirely upon theanatomical extent of disease, was the major determinant of therapy inNHL. This information may be refined by incorporating additionalprognostic pointers, including age, serum lactate dehydrogenase levelsand performance status. Even so, knowledge of the Ann Arbor stagingsystem, together with the histological and immunological subtype of thetumour, is still the major determinant of treatment.

Low grade NHL has an indolent course, with a median patient survival of8 to 10 years. Survival is little impacted by currently availabletherapy, although irradiation of local disease and chemotherapy forsystemic symptoms improves patients' quality of life. Combinationchemotherapy may be reserved for relapsed disease. Intermediate diseaseand, especially, high grade disease is extremely aggressive and tends todisseminate. Disease of this grade requires urgent treatment.Radiotherapy may be a useful component of treatment in patients withvery bulky disease. Many different chemotherapy regimens have beenemployed, and long-term disease-free survival may be obtained in morethan half of patients. High dose therapy with stem cell support wasintroduced initially for patients with relapsed or refractory disease,but is now increasingly finding a place in first line therapy forpatients with poor-risk disease. The tendency in recent years for anincreasingly aggressive therapeutic approach must be balanced againstthe generally elderly age and relative debility of many patients withNHL, and by the need to match the toxicity of treatment to theindividual prognosis of each patient's disease.

Improved treatments, that are more effective and better tolerated, areneeded. Agents recently introduced include new cytotoxic drugs,progressively incorporated into combinations, and the introduction ofantibody-based therapies.

Non-Hodgkin's lymphoma encompasses a range of B cell lymphomas. B cellantigens therefore represent suitable targets for antibody therapy.

CD22 is a 135 kDa membrane glycoprotein belonging to a family of sialicacid binding proteins called sialoadhesins. It is detected in thecytoplasm early in B cell development, appears on the cell surfacesimultaneously with IgD and is found on most mature B cells. Expressionis increased following B cell activation. CD22 is lost with terminaldifferentiation and is generally reported as being absent on plasmacells. Thus this internalising antigen is present on the surface ofpre-B cells and mature B cells but not stem cells or plasma cells.

Two isoforms of CD22 exist in man. The predominant form (CD22β) contains7 immunoglobulin-like (Ig-like) domains in the extracellular region. TheCD22α variant lacks Ig-like domain 4 and may have a truncatedcytoplasmic domain. Antibodies which block CD22 adhesion to monocytes,neutrophils, lymphocytes and erythrocytes have been shown to bind withinthe first or second Ig-like domain.

The cytoplasmic domain of CD22 is tyrosine phosphorylated upon ligationof the B cell antigen receptor and associates with Lyk, Syk andphosphatidyl inositol 3-kinase. The function of CD22 is to down-modulatethe B cell activation threshold. It can also mediate cell adhesionthrough interaction with cells bearing the appropriatesialoglycoconjugates.

CD22 is expressed in most B cell leukaemias and lymphomas, includingNHL, acute lymphoblastic leukaemia (B-ALL), chronic lymphocyticleukaemia (B-CLL) and especially acute non-lymphocytic leukaemia (ANLL).

Monoclonal antibodies against CD22 have been described in the prior art.WO 98/41641 describes recombinant anti-CD22 antibodies with cysteineresidues at V_(H)44 and V_(L)100. WO 96/04925 describes the V_(H) andV_(L) regions of the anti-CD22 antibody LL2. U.S. Pat. No. 5,686,072describes combinations of anti-CD22 and anti-CD19 immunotoxins. WO98/42378 describes the use of naked anti-CD22 antibodies for thetreatment of B-cell malignancies.

A number of antibody-based therapeutics have either been recentlylicensed, eg. Rituxan (an unlabeled chimeric human γ1 (+mγ1V-region)specific for CD20), or are in clinical trials for this disease. Theserely either on complement- or ADCC-mediated killing of B cells or theuse of radionuclides, such as ¹³¹I or ⁹⁰Y, which have associatedpreparation and use problems for clinicians and patients. There is aneed for an antibody molecule to treat NHL which can be used repeatedlyand produced easily and efficiently. There is also a need for anantibody molecule, which has high affinity for CD22 and lowimmunogenicity in humans.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides an antibody moleculehaving specificity for human CD22, comprising a heavy chain wherein thevariable domain comprises a CDR (as defined by Kabat et al., (supra))having the sequence given as H1 in FIG. 1 (SEQ ID NO:1) for CDR-H1, asH2 in FIG. 1 (SEQ ID NO:2) or an H2 from which a potential glycosylationsite has been removed, or an H2 in which the lysine residue at position60 (according to the Kabat numbering system) has been replaced by analternative amino acid, or an H2 in which both the glycosylation siteand the reactive lysine at position 60 have been removed for CDR-H2 oras H3 in FIG. 1 (SEQ ID NO:3) for CDR-H3.

The antibody molecule of the first aspect of the present inventioncomprises at least one CDR selected from H1, H2 and H3 (SEQ ID NO:1, SEQID NO:2 and SEQ ID NO:3) for the heavy chain variable domain.Preferably, the antibody molecule comprises at least two and morepreferably all three CDRs in the heavy chain variable domain.

In a second aspect of the present invention, there is provided anantibody molecule having specificity for human CD22, comprising a lightchain wherein the variable domain comprises a CDR (as defined by Kabatet al., (supra)) having the sequence given as L1 in FIG. 1 (SEQ ID NO:4)for CDR-L1, L2 in FIG. 1 (SEQ ID NO:5) for CDR-L2 or L3 in FIG. 1 (SEQID NO:6) for CDR-L3.

The antibody molecule of the second aspect of the present inventioncomprises at least one CDR selected from L1, L2 and L3 (SEQ ID NO:4; SEQID NO:5 and SEQ ID NO:6) for the light chain variable domain.Preferably, the antibody molecule comprises at least two and morepreferably all three CDRs in the light chain variable domain.

The antibody molecules of the first and second aspects of the presentinvention preferably have a complementary light chain or a complementaryheavy chain, respectively.

Preferably, the antibody molecule of the first or second aspect of thepresent invention comprises a heavy chain wherein the variable domaincomprises a CDR (as defined by Kabat et al., (supra)) having thesequence given as H1 in FIG. 1 (SEQ ID NO:1) for CDR-H1, as H2 in FIG. 1(SEQ ID NO:2) or an H2 from which a potential glycosylation site hasbeen removed, or an H2 in which the lysine residue at position 60(according to the Kabat numbering system) has been replaced by analternative amino acid, or an H2 in which both the glycosylation siteand the reactive lysine at position 60 have been removed for CDR-H2 oras H3 in FIG. 1 (SEQ ID NO:3) for CDR-H3 and a light chain wherein thevariable domain comprises a CDR (as defined by Kabat et al., (supra))having the sequence given as L1 in FIG. 1 (SEQ ID NO:4) for CDR-L1, asL2 in FIG. 1 (SEQ ID NO:5) for CDR-L2 or as L3 in FIG. 1 (SEQ ID NO:6)for CDR-L3.

The CDRs given in SEQ IDS NOs:1 to 6 and in FIG. 1 referred to above arederived from a mouse monoclonal antibody 5/44.

The complete sequences of the variable domains of the mouse 5/44antibody are shown in FIG. 2 (light chain) (SEQ ID NO:7) and FIG. 3(heavy chain) (SEQ ID NO:8). This mouse antibody is also referred tobelow as “the donor antibody” or the “murine monoclonal antibody”.

A first alternatively preferred embodiment of the first or second aspectof the present invention is the mouse monoclonal antibody 5/44 havingthe light and heavy chain variable domain sequences shown in FIG. 2 (SEQID NO:7) and FIG. 3 (SEQ ID NO:8), respectively. The light chainconstant region of 5/44 is kappa and the heavy chain constant region isIgG1.

In a second alternatively preferred embodiment, the antibody accordingto either of the first and second aspects of the present invention is achimeric mouse/human antibody molecule, referred to herein as thechimeric 5/44 antibody molecule. The chimeric antibody moleculecomprises the variable domains of the mouse monoclonal antibody 5/44(SEQ ID NOs:7 and 8) and human constant domains. Preferably, thechimeric 5/44 antibody molecule comprises the human C kappa domain(Hieter et al., Cell, 22, 197-207, 1980; Genebank accession numberJ00241) in the light chain and the human gamma 4 domains (Flanagan etal., Nature, 300, 709-713, 1982) in the heavy chain, optionally with theserine residue at position 241 replaced by a proline residue.

Preferably, the antibody of the present invention comprises a heavychain wherein the variable domain comprises as CDR-H2 (as defined byKabat et al., (supra)) an H2′ in which a potential glycosylation sitesequence has been removed and which unexpectedly increased the affinityof the chimeric 5/44 antibody for the CD22 antigen and which preferablyhas as CDR-H2 the sequence given as H2′ (SEQ ID NO:13).

Alternatively or additionally, the antibody of the present invention maycomprise a heavy chain wherein the variable domain comprises as CDR-H2(as defined by Kabat et al., (supra)) an H2″ in which a lysine residueat position 60, which is located at an exposed position within CDR-H2and which is considered to have the potential to react with conjugationagents resulting in a reduction of antigen binding affinity, issubstituted for an alternative amino acid to result in a conservedsubstitution. Preferably CDR-H2 has the sequence given as H2″ (SEQ IDNO:15).

Alternatively or additionally, the antibody of the present invention maycomprise a heavy chain wherein the variable domain comprises as CDR-H2(as defined by Kabat et al., (supra)) an H2′″ in which both thepotential glycosylation site sequence and the lysine residue at position60, are substituted for alternative amino acids. Preferably CDR-H2 hasthe sequence given as H2′″ (SEQ ID NO:16).

In a third alternatively preferred embodiment, the antibody according toeither of the first and second aspects of the present invention is aCDR-grafted antibody molecule. The term “a CDR-grafted antibodymolecule” as used herein refers to an antibody molecule wherein theheavy and/or light chain contains one or more CDRs (including, ifdesired, a modified CDR) from a donor antibody (e.g. a murine monoclonalantibody) grafted into a heavy and/or light chain variable regionframework of an acceptor antibody (e.g. a human antibody).

Preferably, such a CDR-grafted antibody has a variable domain comprisinghuman acceptor framework regions as well as one or more of the donorCDRs referred to above.

When the CDRs are grafted, any appropriate acceptor variable regionframework sequence may be used having regard to the class/type of thedonor antibody from which the CDRs are derived, including mouse, primateand human framework regions. Examples of human frameworks which can beused in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY andPOM (Kabat et al. (supra)). For example, KOL and NEWM can be used forthe heavy chain, REI can be used for the light chain and EU, LAY and POMcan be used for both the heavy chain and the light chain. Alternatively,human germline sequences may be used. The preferred framework region forthe light chain is the human germline sub-group sequence (DPK9+JK1)shown in FIG. 5 (SEQ ID NO:17). The preferred framework region for theheavy chain is the human sub-group sequence (DP7+JH4) shown in FIG. 6(SEQ ID NO:21).

In a CDR-grafted antibody of the present invention, it is preferred touse as the acceptor antibody one having chains which are homologous tothe chains of the donor antibody. The acceptor heavy and light chains donot necessarily need to be derived from the same antibody and may, ifdesired, comprise composite chains having framework regions derived fromdifferent chains.

Also, in a CDR-grafted antibody of the present invention, the frameworkregions need not have exactly the same sequence as those of the acceptorantibody. For instance, unusual residues may be changed to morefrequently-occurring residues for that acceptor chain class or type.Alternatively, selected residues in the acceptor framework regions maybe changed so that they correspond to the residue found at the sameposition in the donor antibody or to a residue that is a conservativesubstitution for the residue found at the same position in the donorantibody. Such changes should be kept to the minimum necessary torecover the affinity of the donor antibody. A protocol for selectingresidues in the acceptor framework regions which may need to be changedis set forth in WO 91/09967.

Preferably, in a CDR-grafted antibody molecule according to the presentinvention, if the acceptor light chain has the human sub-group DPK9+JK1sequence (shown in FIG. 5) (SEQ ID NO:17 (DPK9) plus SEQ ID NO:18 (JK1))then the acceptor framework regions of the light chain comprise donorresidues at positions 2, 4, 37, 38, 45 and 60 and may additionallycomprise a donor residue at position 3 (according to Kabat et al.(supra)).

Preferably, in a CDR-grafted antibody molecule of the present invention,if the acceptor heavy chain has the human DP7+JH4 sequence (shown inFIG. 6) (SEQ ID NO:21 (DP7) plus SEQ ID NO:22 (JH4)), then the acceptorframework regions of the heavy chain comprise, in addition to one ormore donor CDRs, donor residues at positions 1, 28, 48, 71 and 93 andmay additionally comprise donor residues at positions 67 and 69(according to Kabat et al. (supra)).

Donor residues are residues from the donor antibody, i.e. the antibodyfrom which the CDRs were originally derived.

Preferably, the antibody of the present invention comprises a heavychain wherein the variable domain comprises as CDR-H2 (as defined byKabat et al., (supra)) an H2′ in which a potential glycosylation sitesequence has been removed in order to increase the affinity of thechimeric 5/44 antibody for the CD22 antigen and which preferably has asCDR-H2 the sequence given as H2′ (SEQ ID NO:13).

Alternatively or additionally, the antibody of the present invention maycomprise a heavy chain wherein the variable domain comprises as CDR-H2(as defined by Kabat et al., (supra)) an H2″ in which a lysine residueat position 60, which is located at an exposed position within CDR-H2and which is considered to have the potential to react with conjugationagents resulting in a reduction of antigen binding affinity, issubstituted for an alternative amino acid. Preferably CDR-H2 has thesequence given as H2″ (SEQ ID NO:15).

Alternatively or additionally, the antibody of the present invention maycomprise a heavy chain wherein the variable domain comprises as CDR-H2(as defined by Kabat et al., (supra)) an H2′″ in which both thepotential glycosylation site sequence and the lysine residue at position60, are substituted for alternative amino acids. Preferably CDR-H2 hasthe sequence given as H2′″ (SEQ ID NO:16).

The antibody molecule of the present invention may comprise: a completeantibody molecule, having full length heavy and light chains; a fragmentthereof, such as a Fab, modified Fab, Fab′, F(ab′)₂ or Fv fragment; alight chain or heavy chain monomer or dimer; a single chain antibody,e.g. a single chain Fv in which the heavy and light chain variabledomains are joined by a peptide linker. Similarly, the heavy and lightchain variable regions may be combined with other antibody domains asappropriate.

The antibody molecule of the present invention may have an effector or areporter molecule attached to it. For instance, it may have amacrocycle, for chelating a heavy metal atom, or a toxin, such as ricin,attached to it by a covalent bridging structure. Alternatively,procedures of recombinant DNA technology may be used to produce anantibody molecule in which the Fc fragment (CH2, CH3 and hinge domains),the CH2 and CH3 domains or the CH3 domain of a complete immunoglobulinmolecule has (have) been replaced by, or has (have) attached thereto bypeptide linkage, a functional non-immunoglobulin protein, such as anenzyme or toxin molecule.

The antibody molecule of the present invention preferably has a bindingaffinity of at least 0.85×10⁻¹⁰ M, more preferably at least 0.75×10⁻¹⁰ Mand most preferably at least 0.5×10⁻¹⁰ M.

Preferably, the antibody molecule of the present invention comprises thelight chain variable domain 5/44-gL1 (SEQ ID NO:19) and the heavy chainvariable domain 5/44-gH7 (SEQ ID NO:27). The sequences of the variabledomains of these light and heavy chains are shown in FIGS. 5 and 6,respectively.

The present invention also relates to variants of the antibody moleculeof the present invention, which have an improved affinity for CD22. Suchvariants can be obtained by a number of affinity maturation protocolsincluding mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403,1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783,1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol.,250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin.Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol.Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391,288-291, 1998). Vaughan et al. (supra) discusses these methods ofaffinity maturation.

The present invention also provides a DNA sequence encoding the heavyand/or light chain(s) of the antibody molecule of the present invention.

Preferably, the DNA sequence encodes the heavy or the light chain of theantibody molecule of the present invention.

The DNA sequence of the present invention may comprise synthetic DNA,for instance produced by chemical processing, cDNA, genomic DNA or anycombination thereof.

The present invention also relates to a cloning or expression vectorcomprising one or more DNA sequences of the present invention.Preferably, the cloning or expression vector comprises two DNAsequences, encoding the light chain and the heavy chain of the antibodymolecule of the present invention, respectively.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

DNA sequences which encode the antibody molecule of the presentinvention can be obtained by methods well known to those skilled in theart. For example, DNA sequences coding for part or all of the antibodyheavy and light chains may be synthesised as desired from the determinedDNA sequences or on the basis of the corresponding amino acid sequences.

DNA coding for acceptor framework sequences is widely available to thoseskilled in the art and can be readily synthesised on the basis of theirknown amino acid sequences.

Standard techniques of molecular biology may be used to prepare DNAsequences coding for the antibody molecule of the present invention.Desired DNA sequences may be synthesised completely or in part usingoligonucleotide synthesis techniques. Site-directed mutagenesis andpolymerase chain reaction (PCR) techniques may be used as appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecule of the present invention.Bacterial, for example E. coli, and other microbial systems may be used,in part, for expression of antibody fragments such as Fab and F(ab′)₂fragments, and especially Fv fragments and single chain antibodyfragments, for example, single chain Fvs. Eukaryotic, e.g. mammalian,host cell expression systems may be used for production of largerantibody molecules, including complete antibody molecules. Suitablemammalian host cells include CHO, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell containing a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

The present invention also provides a therapeutic or diagnosticcomposition comprising an antibody molecule of the present invention incombination with a pharmaceutically acceptable excipient, diluent orcarrier.

The present invention also provides a process for preparation of atherapeutic or diagnostic composition comprising admixing the antibodymolecule of the present invention together with a pharmaceuticallyacceptable excipient, diluent or carrier.

The antibody molecule may be the sole active ingredient in thetherapeutic or diagnostic composition or may be accompanied by otheractive ingredients including other antibody ingredients, for exampleanti-T cell, anti-IFNγ or anti-LPS antibodies, or non-antibodyingredients such as xanthines.

The pharmaceutical compositions preferably comprise a therapeuticallyeffective amount of the antibody of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any antibody, the therapeutically effectivedose can be estimated initially either in cell culture assays or inanimal models, usually in rodents, rabbits, dogs, pigs or primates. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The precise effective amount for a human subject will depend upon theseverity of the disease state, the general health of the subject, theage, weight and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities andtolerance/response to therapy. This amount can be determined by routineexperimentation and is within the judgement of the clinician. Generally,an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.1mg/kg to 20 mg/kg, more preferably about 15 mg/kg.

Compositions may be administered individually to a patient or may beadministered in combination with other agents, drugs or hormones.

The dose at which the antibody molecule of the present invention isadministered depends on the nature of the condition to be treated, thegrade of the malignant lymphoma or leukaemia and on whether the antibodymolecule is being used prophylactically or to treat an existingcondition.

The frequency of dose will depend on the half-life of the antibodymolecule and the duration of its effect. If the antibody molecule has ashort half-life (e.g. 2 to 10 hours) it may be necessary to give one ormore doses per day. Alternatively, if the antibody molecule has a longhalf life (e.g. 2 to 15 days) it may only be necessary to give a dosageonce per day, once per week or even once every 1 or 2 months.

A pharmaceutical composition may also contain a pharmaceuticallyacceptable carrier for administration of the antibody. The carriershould not itself induce the production of antibodies harmful to theindividual receiving the composition and should not be toxic. Suitablecarriers may be large, slowly metabolised macromolecules such asproteins, polypeptides, liposomes, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers andinactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Preferred forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilising and/or dispersing agents.Alternatively, the antibody molecule may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals.However, it is preferred that the compositions are adapted foradministration to human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.Typically, the therapeutic compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule. As such, it will be susceptible todegradation in the gastrointestinal tract. Thus, if the composition isto be administered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

It is also envisaged that the antibody of the present invention will beadministered by use of gene therapy. In order to achieve this, DNAsequences encoding the heavy and light chains of the antibody moleculeunder the control of appropriate DNA components are introduced into apatient such that the antibody chains are expressed from the DNAsequences and assembled in situ.

The present invention also provides the antibody molecule of the presentinvention for use in treating a disease mediated by cells expressingCD22.

The present invention further provides the use of the antibody moleculeaccording to the present invention in the manufacture of a medicamentfor the treatment of a disease mediated by cells expressing CD22.

The antibody molecule of the present invention may be utilised in anytherapy where it is desired to reduce the level of cells expressing CD22that are present in the human or animal body. These CD22-expressingcells may be circulating in the body or be present in an undesirablyhigh level localised at a particular site in the body. For example,elevated levels of cells expressing CD22 will be present in B celllymphomas and leukaemias. The antibody molecule of the present inventionmay be utilised in the therapy of diseases mediated by cells expressingCD22.

The antibody molecule of the present invention is preferably used fortreatment of malignant lymphomas and leukaemias, most preferably NHL.

The present invention also provides a method of treating human or animalsubjects suffering from or at risk of a disorder mediated by cellsexpressing CD22, the method comprising administering to the subject aneffective amount of the antibody molecule of the present invention.

The antibody molecule of the present invention may also be used indiagnosis, for example in the in vivo diagnosis and imaging of diseasestates involving cells that express CD22.

The present invention is further described by way of illustration onlyin the following examples, which refer to the accompanying Figures, inwhich:

FIG. 1 shows the amino acid sequence of the CDRs of mouse monoclonalantibody 5/44 (SEQ ID NOs:1 to 6);

FIG. 2 shows the complete sequence of the light chain variable domain ofmouse monoclonal antibody 5/44 (nucleotide sequence-SEQ ID NO:48; aminoacid sequence-SEQ ID NO: 7); antisense nucleotide strand-SEQ ID NO:67;

FIG. 3 shows the complete sequence of the heavy chain variable domain ofmouse monoclonal antibody 5/44 (nucleotide sequence—SEQ ID NO:49; aminoacid sequence-SEQ ID NO:8); antisense nucleotide strand-SEQ ID NO:68;

FIG. 4 shows the strategy for removal of the glycosylation site andreactive lysine in CDR-H2 (SEQ ID NOs:9-12);

FIG. 5 shows the graft design for the 5/44 light chain sequence(V_(L)-SEQ ID NO:7; DPK9-SEQ ID NO:17, SEQ ID NO:69, and SEQ ID NO:70,respectively; JK1-SEQ ID NO:18 gL1-SEQ ID NO:19; and gL2-SEQ ID NO:20);

FIG. 6 shows the graft design for the 5/44 heavy chain sequence(V_(H)-SEQ ID NO:8, DP7-SEQ ID NO:24, gH5-SEQ ID NO:25, gH6-SEQ IDNO:26, gH7-SEQ ID NO:27, and JH4-SEQ ID NO:22);

FIGS. 7A-7B show the vectors pMRR14 and pMRR10.1;

FIG. 8 shows the Biacore assay results of the chimeric 5/44 mutants;

FIG. 9 shows the oligonucleotides for 5/44 gH1 (SEQ ID NOs:32-39,respectively) and gL1 (SEQ ID NOs:40-47, respectively) gene assemblies;

FIGS. 10A-10B show the intermediate vectors pCR2.1(544gH1) andpCR2.1(544gL1);

FIG. 11 shows the oligonucleotide cassettes used to make further grafts(gH4-SEQ ID NOs:52, 53, and 62, respectively, gH5—SEQ ID NOs:54, 55, and63, respectively; gH6—SEQ ID NOs:56, 57, and 64, respectively; gH7—SEQID NOs: 58, 59, and 65, respectively; and gL2—SEQ ID NOs:60, 61, and 66,respectively;

FIGS. 12A-12B show the competition assay between fluorescently labelledmouse 5/44 antibody and grafted variants; and

FIG. 13 shows the full DNA and protein sequence of the grafted heavy andlight chains—a) SEQ ID NO:30 (amino acid), SEQ ID NO:31 (nucleotide),and SEQ ID NO:63 (antisense nucleotide strand); b) SEQ ID NO: 28 (aminoacid), SEQ ID NO:29 (nucleotide), and SEQ ID NO:74 (antisense strand).

DETAILED DESCRIPTION OF THE INVENTION Example 1: Generation of CandidateAntibodies

A panel of antibodies against CD22 were selected from hybridomas usingthe following selection criteria: binding to Daudi cells,internalisation on Daudi cells, binding to peripheral blood mononuclearcells (PBMC), internalisation on PBMC, affinity (greater than 10⁻⁹M),mouse γ1 and production rate. 5/44 was selected as the preferredantibody.

Example 2: Gene Cloning and Expression of a Chimeric 5/44 AntibodyMolecule

Preparation of 5/44 Hybridoma Cells and RNA Preparation Therefrom

Hybridoma 5/44 was generated by conventional hybridoma technologyfollowing immunisation of mice with human CD22 protein. RNA was preparedfrom 5/44 hybridoma cells using a RNEasy kit (Qiagen, Crawley, UK;Catalogue No. 74106). The RNA obtained was reverse transcribed to cDNA,as described below.

Distribution of CD22 on NHL Tumours

An immunohistochemistry study was undertaken to examine the incidenceand distribution of staining using the 5/44 anti-CD22 monoclonalantibodies. Control anti-CD20 and anti-CD79a antibodies were included inthe study to confirm B cell areas of tumours.

A total of 50 tumours were studied and these were categorised as followsby using the Working Formulation and REAL classification systems:

7 B lymphoblastic leukaemia/lymphoma (High/l)

4 B-CLL/small lymphocytic lymphoma (Low/A)

3 lymphoplasmacytoid/Immunocytoma (Low/A)

1 Mantle cell (Int/F)

14 Follicle center lymphoma (Low to Int/D)

13 Diffuse large cell lymphoma (Int to High/G,H)

6 Unclassifiable (K)

2 T cell lymphomas

40 B cell lymphomas were positive for CD22 antigen with the 5/44antibody at 0.1 μg/ml and a further 6 became positive when theconcentration was increased to 0.5 μg/ml. For the remaining 2 B celltumours that were negative at 0.1 μg/ml, there was insufficient tissueremaining to test at the higher concentration. However, parallel testingwith another Celltech anti-CD22 antibody 6/13, which gave strongerstaining than 5/44, resulted in all 48 B cell lymphomas stainingpositive for CD22.

Thus, it is possible to conclude that the CD22 antigen is widelyexpressed on B cell lymphomas and thus provides a suitable target forimmunotherapy in NHL.

PCR Cloning of 5/44 V_(H) and V_(L)

cDNA sequences coding for the variable domains of 5/44 heavy and lightchains were synthesised using reverse transcriptase to produce singlestranded cDNA copies of the mRNA present in the total RNA. This was thenused as the template for amplification of the murine V-region sequencesusing specific oligonucleotide primers by the Polymerase Chain Reaction(PCR).

a) cDNA Synthesis

cDNA was synthesised in a 20 μl reaction volume containing the followingreagents: 50 mM Tris-HCl pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mMMgCl₂, 0.5 mM each deoxyribonucleoside triphosphate, 20 units RNAsin, 75ng random hexanucleotide primer, 2 μg 5/44 RNA and 200 units MoloneyMurine Leukemia Virus reverse transcriptase. After incubation at 42° C.for 60 minutes, the reaction was terminated by heating at 95° C. for 5minutes.

b) PCR

Aliquots of the cDNA were subjected to PCR using combinations of primersspecific for the heavy and light chains. Degenerate primer poolsdesigned to anneal with the conserved sequences of the signal peptidewere used as forward primers. These sequences all contain, in order, arestriction site (V_(L) SfuI; V_(H) HindIII) starting 7 nucleotides fromtheir 5′ ends, the sequence GCCGCCACC (SEQ ID NO:50), to allow optimaltranslation of the resulting mRNAs, an initiation codon and 20-30nucleotides based on the leader peptide sequences of known mouseantibodies (Kabat et al., Sequences of proteins of immunologicalinterest, 5^(th) Edition, 1991, U.S. Department of Health and HumanServices, Public Health Service, National Institutes of Health).

The 3′ primers are designed to span the framework 4 J-C junction of theantibody and contain a restriction site for the enzyme BsiWI tofacilitate cloning of the V_(L) PCR fragment. The heavy chain 3′ primersare a mixture designed to span the J-C junction of the antibody. The 3′primer includes an ApaI restriction site to facilitate cloning. The 3′region of the primers contains a mixed sequence based on those found inknown mouse antibodies (Kabat et al., 1991, supra).

The combinations of primers described above enable the PCR products forV_(H) and V1 to be cloned directly into an appropriate expression vector(see below) to produce chimeric (mouse-human) heavy and light chains andfor these genes to be expressed in mammalian cells to produce chimericantibodies of the desired isotype.

Incubations (100 μl) for the PCR were set up as follows. Each reactioncontained 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.01% w/vgelatin, 0.25 mM each deoxyribonucleoside triphosphate, 10 pmoles 5′primer mix, 10 pmoles 3′ primer, 1 cDNA and 1 unit Taq polymerase.Reactions were incubated at 95° C. for 5 minutes and then cycled through94° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1 minute. After30 cycles, aliquots of each reaction were analysed by electrophoresis onan agarose gel.

For the heavy chain V-region, an amplified DNA product was only obtainedwhen a primer pool annealing within the start of framework I replacedthe signal peptide primer pool. The fragments were cloned into DNAsequencing vectors. The DNA sequence was determined and translated togive a deduced amino acid sequence. This deduced sequence was verifiedby reference to the N-terminal protein sequence determinedexperimentally. FIGS. 2 and 3 shows the DNA/protein sequence of themature light and heavy chain V-regions of mouse monoclonal 5/44respectively.

c) Molecular Cloning of the PCR Fragments

The murine v-region sequences were then cloned into the expressionvectors pMRR10.1 and pMRR14 (FIG. 7). These are vectors for theexpression of light and heavy chain respectively containing DNA encodingconstant regions of human kappa light chain and human gamma-4 heavychain. The V_(L) region was sub-cloned into the expression vector byrestriction digest and ligation from the sequencing vector, using SfuIand BsiWI restriction sites, creating plasmid pMRR10(544cL). The heavychain DNA was amplified by PCR using a 5′ primer to introduce a signalpeptide, since this was not obtained in the cloning strategy—a mouseheavy chain antibody leader from a different in-house hybridoma (termed162) was employed. The 5′ primer had the following sequence:

(SEQ ID NO: 51) ^(5′)GCGCGCAAGCTTGCCGCCACCATGGACTTCGGATTCTCTCTCGTGTTCCTGGCACTCATTCTCAAGGGAGTGCAGTGTGAGGTGCAGCTCGTCGA GTCTGG^(3′).

The reverse primer was identical to that used in the original V_(H) genecloning. The resultant PCR product was digested with enzymes HindIII andApaI, was sub-cloned, and its DNA sequence was confirmed, creatingplasmid pMRR14(544cH). Transient co-transfection of both expressionvectors into CHO cells generated chimeric c5/44 antibody. This wasachieved using the Lipofectamine reagent according to the manufacturer'sprotocols (InVitrogen: Life Technology, Groningen, The Netherlands.Catalogue no. 11668-027).

Removal of Glycosylation Site and Reactive Lysine

A potential N-linked glycosylation site sequence was observed in CDR-H2,having the amino acid sequence N-Y-T (FIG. 3). SDS-PAGE, Westernblotting and carbohydrate staining of gels of 5/44 and its fragments(including Fab) indicated that this site was indeed glycosylated (notshown). In addition, a lysine residue was observed at an exposedposition within CDR-H2, which had the potential to reduce the bindingaffinity of the antibody by providing an additional site for conjugationwith an agent with which the antibody may be conjugated.

A PCR strategy was used to introduce amino acid substitutions into theCDR-H2 sequence in an attempt to remove the glycosylation site and/orthe reactive lysine, as shown in FIG. 4. Forward primers encoding themutations N55Q, T57A or T57V were used to remove the glycosylation site(FIG. 4) and a fourth forward primer containing the substitution K60R,was generated to remove the reactive lysine residue (FIG. 4). Aframework 4 reverse primer was used in each of these PCR amplifications.The PCR products were digested with the enzymes XbaI and ApaI and wereinserted into pMRR14(544cH) (also cleaved with XbaI and ApaI) togenerate expression plasmids encoding these mutants. The N55Q, T57A andT57V mutations ablate the glycosylation site by changing the amino acidsequence away from the consensus N-X-T/S whilst the K60R mutationreplaces the potentially reactive lysine with the similarly positivelycharged residue arginine. The resultant cH variant plasmids wereco-transfected with the cL plasmid to generate expressed chimericantibody variants.

Evaluation of Activities of Chimeric Genes

The activities of the chimeric genes were evaluated following transienttransfection into CHO cells.

c) Determination of Affinity Constants by BiaCore Analysis.

The affinities of chimeric 5/44 or its variants, which have had theirglycosylation site or their reactive lysine removed, were investigatedusing BIA technology for binding to CD22-mFc constructs. The results areshown in FIG. 8. All binding measurements were performed in the BIAcore™2000 instrument (Pharmacia Biosensor AB, Uppsala, Sweden). The assay wasperformed by capture of CD22mFc via the immobilised anti-mouse Fc. Theantibody was in the soluble phase. Samples, standard, and controls (50ul) were injected over immobilised anti-mouse Fc followed by antibody inthe soluble phase. After each cycle the surface was regenerated with 50ul of 40 mM HCl at 30 ul/min. The kinetic analysis was performed usingthe BIAevaluation 3.1 software (Pharmacia).

Removal of the glycosylation site in construct T57A resulted in aslightly faster on-rate and a significantly slower off-rate compared tothe chimeric 5/44, giving an affinity improvement of approximately5-fold. The N55Q mutation had no effect on affinity. This result wasunexpected as it suggests that the removal of the carbohydrate itselfapparently has no effect on binding (as with the N55Q change). Theimproved affinity was observed only with the T57A change. One possibleexplanation is that, regardless of the presence of carbohydrate, thethreonine at position 57 exerts a negative effect on binding that isremoved on conversion of threonine to alanine. The hypothesis that thesmall size of alanine is important, and that the negative effect ofthreonine is related to its size, is supported from the result obtainedusing the T57V mutation: that replacement with valine at position 57 isnot beneficial (results not shown).

Removal of the lysine by the K60R mutation had a neutral effect onaffinity, i.e. the introduction of arginine removes a potential reactivesite without compromising affinity.

The mutations for removal of the glycosylation site and for removal ofthe reactive lysine were therefore both included in the humanisationdesign.

Example 2: CDR-Grafting of 5/44

The molecular cloning of genes for the variable regions of the heavy andlight chains of the 5/44 antibody and their use to produce chimeric(mouse/human) 5/44 antibodies has been described above. The nucleotideand amino acid sequences of the mouse 5/44 V_(L) and V_(H) domains areshown in FIGS. 2 and 3 (SEQ ID NOs:7 and 8), respectively. This exampledescribes the CDR-grafting of the 5/44 antibody onto human frameworks toreduce potential immunogenicity in humans, according to the method ofAdair et al., (WO91/09967).

CDR-Grafting of 5/44 Light Chain

Protein sequence alignment with consensus sequences from human sub-groupI kappa light chain V region indicated 64% sequence identity.Consequently, for constructing the CDR-grafted light chain, the acceptorframework regions chosen corresponded to those of the human VK sub-groupI germline 012,DPK9 sequence. The framework 4 acceptor sequence wasderived from the human J-region germline sequence JK1.

A comparison of the amino acid sequences of the framework regions ofmurine 5/44 and the acceptor sequence is given in FIG. 5 and shows thatthere are 27 differences between the donor and acceptor chains. At eachposition, an analysis was made of the potential of the murine residue tocontribute to antigen binding, either directly or indirectly, througheffects on packing or at the V_(H)/V_(L) interface. If a murine residuewas considered important and sufficiently different from the humanresidue in terms of size, polarity or charge, then that murine residuewas retained. Based on this analysis, two versions of the CDR-graftedlight chain, having the sequences given in SEQ ID NO:19 and SEQ ID NO:20(FIG. 5), were constructed.

CDR-Grafting of 5/44 Heavy Chain

CDR-grafting of 5/44 heavy chain was accomplished using the samestrategy as described for the light chain. The V-domain of 5/44 heavychain was found to be homologous to human heavy chains belonging tosub-group I (70% sequence identity) and therefore the sequence of thehuman sub-group I germline framework VH1-3,DP7 was used as an acceptorframework. The framework 4 acceptor sequences were derived from humanJ-region germline sequence JH4.

A comparison of 5/44 heavy chain with the framework regions is shown inFIG. 6 where it can be seen that the 5/44 heavy chain differs from theacceptor sequence at 22 positions. Analysis of the contribution that anyof these might make to antigen binding led to 5 versions of theCDR-grafted heavy chains being constructed, having the sequences givenin SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ IDNO:27 (FIG. 6).

Construction of Genes for Grafted Sequences.

Genes were designed to encode the grafted sequences gH1 and gL1, and aseries of overlapping oligonucleotides were designed and constructed(FIG. 9). A PCR assembly technique was employed to construct theCDR-grafted V-region genes. Reaction volumes of 100 ul were set upcontaining 10 mM Tris-HCl pH8.3, 1.5 mM MgCl2, 50 mM KCl, 0.001%gelatin, 0.25 mM each deoxyribonucleoside triphosphate, 1 pmole each ofthe ‘internal’ primers (T1, T2, T3, B1, B2, B3), 10 pmole each of the‘external’ primers (F1, R1), and 1 unit of Taq polymerase (AmpliTaq,Applied BioSystems, catalogue no. N808-0171). PCR cycle parameters were94° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1 minute, for 30cycles. The reaction products were then run on a 1.5% agarose gel,excised and recovered using QIAGEN® spin columns (QIAquick® gelextraction kit, cat no. 28706). The DNA was eluted in a volume of 30 μl.Aliquots (1 μl) of the gH1 and gL1 DNA were then cloned into theInVitrogen TOPO® TA cloning vector pCR2.1 TOPO® (catalogue no. K4500-01)according to the manufacturer's instructions. This non-expression vectorserved as a cloning intermediate to facilitate sequencing of a largenumber of clones. DNA sequencing using vector-specific primers was usedto identify correct clones containing gH1 and gL1, creating plasmidspCR2.1 (544gH1) and pCR2.1(544gL1) (FIG. 10).

An oligonucleotide cassette replacement method was used to create thehumanised grafts gH4,5,6 and 7, and gL2. FIG. 11 shows the design of theoligonucleotide cassettes. To construct each variant, the vector(pCR2.1(544gH1) or pCR2.1(544gL1)) was cut with the restriction enzymesshown (XmaI/SacII for the heavy chain, XmaI/BstEII for the light chain).The large vector fragment was gel purified from agarose and was used inligation with the oligonucleotide cassette. These cassettes are composedof 2 complementary oligonucleotides (shown in FIG. 11), mixed at aconcentration of 0.5 pmoles/μ1 in a volume of 200 μl 12.5 mM Tris-HCl pH7.5, 2.5 mM MgCl₂, 25 mM NaCl, 0.25 mM dithioerythritol. Annealing wasachieved by heating to 95° C. for 3 minutes in a waterbath (volume 500ml) then allowing the reaction to slow-cool to room temperature. Theannealed oligonucleotide cassette was then diluted ten-fold in waterbefore ligation into the appropriately cut vector. DNA sequencing wasused to confirm the correct sequence, creating plasmids pCR2.1(5/44-gH4-7) and pCR2.1(5/44-gL2). The verified grafted sequences werethen sub-cloned into the expression vectors pMRR14 (heavy chain) andpMR10.1 (light chain).

CD22 Binding Activity of CDR-Grafted Sequences

The vectors encoding grafted variants were co-transfected into CHO cellsin a variety of combinations, together with the original chimericantibody chains. Binding activity was compared in a competition assay,competing the binding of the original mouse 5/44 antibody for binding toRamos cells (obtained from ATCC, a Burkitt's lymphoma lymphoblast humancell line expressing surface CD22). This assay was considered the bestway to compare grafts in their ability to bind to cell surface CD22. Theresults are shown in FIG. 8. As can be seen, there is very littledifference between any of the grafts, all performing more effectivelythan the chimeric at competing against the murine parent. Theintroduction of the 3 additional human residues at the end of CDR H2(gH6 and gH7) does not appear to have affected binding.

The graft combination with the least number of murine residues wasselected, gL1gH7. The light chain graft gL1 has 6 donor residues.Residues V2, V4, L37 and Q45 are potentially important packing residues.Residue H38 is at the V_(H)/V_(L) interface. Residue D60 is a surfaceresidue close to the CDR-L2 and may directly contribute to antigenbinding. Of these residues, V2, L37, Q45 and D60 are found in germlinesequences of human kappa genes from other sub-groups. The heavy chaingraft gH7 has 4 donor framework residues (Residue R28 is considered tobe part of CDR-H1 under the structural definition used in CDR-grafting(se Adair et al (1991 WO91/09967)). Residues E1 and A71 are surfaceresidues close to the CDR's. Residue I48 is a potential packing residue.Residue T93 is present at the V_(H)/V_(L) interface. Of these residues,E1 and A71 are found in other germline genes of human sub-group I.Residue I48 is found in human germline sub-group 4, and T73 is found inhuman germline sub-group 3.

The full DNA and protein sequence of both the light chain and heavychain, including approximate position of introns within the constantregion genes provided by the vectors, are shown in FIG. 13 and are givenin SEQ ID NO:29 and SEQ ID NO:28 respectively for the light chain andSEQ ID NO:31 and SEQ ID NO:30 respectively for the heavy chain.

DNA encoding these light and heavy chain genes was excised from thesevectors. Heavy chain DNA was digested at the 5′ HindIII site, then wastreated with the Klenow fragment of E. coli DNA polymerase I to create a5′ blunt end. Cleavage at the 3′ EcoRI site resulted in the heavy chainfragment which was purified from agarose gels. In the same way, a lightchain fragment was produced, blunted at the 5′ SfuI site and with a 3′EcoRI site. Both fragments were cloned into DHFR based expressionvectors and used to generate stable cell lines in CHO cells.

All references and patents cited herein are hereby incorporated byreference in their entireties.

What is claimed is:
 1. An antibody molecule that binds human CD22comprising a heavy chain and a light chain, wherein each chain comprisesthree complementarity determining regions (CDRs), wherein CDR-H1comprises the amino acid sequence of SEQ ID NO:1; CDR-H3 comprises theamino acid sequence of SEQ ID NO:3; CDR-L1 comprises the amino acidsequence of SEQ ID NO:4; CDR-L2 comprises the amino acid sequence of SEQID NO:5; and CDR-L3 comprises the amino acid sequence of SEQ ID NO:6. 2.The antibody molecule of claim 1, which is a CDR-grafted antibodymolecule.
 3. The antibody molecule of claim 2, wherein the variabledomain comprises human acceptor framework regions and non-human donorCDRs.
 4. A composition comprising the antibody molecule of claim
 1. 5.The composition according to claim 4, comprising a pharmaceuticallyacceptable excipient, diluent, or carrier.
 6. The composition accordingto claim 4, additionally comprising anti-T cell, anti-IFNγ, anti-LPSantibodies, or a non-antibody ingredient.